General  |   Fertilization  |   Embroy Development

General

Case: 36 year old G0 with a history of subfertility, regular menstrual intervals every 28-29 x 3-5 days, a normal hormone evaluation (TSH and prolactin), an encouraging ovarian reserve (using basal FSH and estradiol concentrations), a normal hysterosalpingogram (normal uterine cavity with bilateral tubal patency), a normal postcoital test, and a husband with unproven fertility and a normal semen analysis.

Question: What should be considered given this information?

Answer: In these situations in which the basic infertility evaluation is normal, I suggest a pelvic evaluation (laparoscopy and hysteroscopy) to identify and treat pathology within the pelvic cavity. The abnormalities most often identified at pelvic evaluation include endometriosis, pelvic adhesions and endometrial polyps (polypoid overgrowths of endometrium that may be found to be “polyps” or simply tufts of endometrium on pathology examination). In my own practice, I find some pelvic abnormalities in about 75% of women with this history (basic noninvasive infertility evaluation is normal).

Looking at this situation in another way may help some couples see the value of the pelvic evaluation. If the eggs are thought to be normal given a history of regular menstrual intervals, a consistent amount of menstrual flow, premenstrual symptomatology, a normal hormone evaluation, and a good ovarian reserve then ovulation induction (to treat an ovulation issue) is not generally suggested. If the sperm appears to be normal on semen analysis, then intrauterine inseminations (to treat a male factor) are not generally suggested. If treatments for ovulation dysfunctions or male factors are not being suggested, then this suggests that there may be additional utility in further identifying and treating a pelvic factor. Some pelvic factor issues have been ruled out with the postcoital test and the hysterosalpingogram but other remain and should be assessed and treated with a pelvic evaluation. If the pelvic evaluation is completely normal, then proceeding to treatments for “unexplained infertility” make most sense.

Case: 28 year old G0 with a history of subfertility, regular menstrual intervals every 29-31 x 5-6 days, a normal hormone evaluation (TSH and prolactin), a mildly abnormal hysterosalpingogram (normal uterine cavity with bilateral tubal patency yet the left tube appears to be slightly dilated along the ampullary and distal portions and there is also a suggestion of peritubal adhesions), a normal postcoital test, and a husband with unproven fertility and a normal semen analysis.

Question: What should be considered given this information?

Answer: In this case, the diagnostic testing has been normal except for some “soft findings” during the hysterosalpingogram (HSG). If the HSG findings were thought to be essentially normal, then a pelvic evaluation would be appropriate to assess and possibly treat a previously unrecognized pelvic issue. If the HSG findings are thought to be significant (suggest pelvic pathology), then a pelvic evaluation is also appropriate to assess the tubes and pelvis and treat any (repairable) abnormalities that are found.

If this woman has any prior history of infection within her abdomen or pelvis, such as appendicitis or pelvic inflammatory disease, then I would most likely give prophylactic antibiotics to reduce the chance of reactivating a “dormant” pelvic infection during pelvic evaluation.

Case: 34 year old G0 with a history of subfertility, regular menstrual intervals every 27-28 x 4-5 days, a normal hormone evaluation (TSH and prolactin), an encouraging ovarian reserve (using basal FSH and estradiol concentrations), a mildly abnormal hysterosalpingogram (irregular borders of the uterine cavity without discrete filling defects with bilateral tubal patency), an abnormal postcoital test (with 0-1 motile sperm per high power field), and a husband with unproven fertility and a normal semen analysis.

Question: What should be considered given this information?

Answer: I generally suggest the least aggressive treatment alternative that has a reasonable chance of success for couples in my own infertility practice. If the less aggressive alternative is ineffective (does not result in a pregnancy) within a reasonable period of time, then I usually suggest more aggressive treatment alternatives. This is often considered a relatively “traditional approach.”

Many infertility practices have a more “contemporary approach” to infertility treatment. The more contemporary approach appears to suggest In Vitro Fertilization (IVF) very early on. The preliminary evaluation determines ovarian reserve, semen quality and a hysterosalpingogram (HSG). If this workup is encouraging, then the couple may consider IVF as the initial treatment. On the one hand, IVF does have a very high success rate per attempted cycle (the “ongoing” pregnancy rate following identification of an active fetal heart in the uterus may be greater than 50% per cycle of IVF). On the other hand, IVF is often very expensive, time consuming and involves some personal risk. Therefore, suggesting IVF as an initial treatment (when other options are available and have not been attempted) seems aggressive to me.

In this case, the postcoital test was abnormal and the HSG was a bit irregular. I would generally suggest a few (3-6) cycles of timed natural (spontaneous) cycle intrauterine insemination (IUI) and if ineffective then consideration of more aggressive options. If the timed IUIs do not result in pregnancy, I generally suggest looking for a (previously unidentified) pelvic factor with a pelvic evaluation (laparoscopy and hysteroscopy) and then reconsidering controlled ovarian hyperstimulation (using menotropins) with IUI.

Fertilization

Case: 30 year old G0 with a history of subfertility, regular menstrual intervals every 27-29 x 3-5 days, a normal hormone evaluation (TSH and prolactin), an encouraging ovarian reserve (using basal FSH and estradiol concentrations), a normal hysterosalpingogram (normal uterine cavity with bilateral tubal patency), a normal postcoital test, a husband with unproven fertility and a normal semen analysis, status post pelvic evaluation (laparoscopy and hysteroscopy) that did not reveal any abnormalities, status post 3 cycles of controlled ovarian hyperstimulation (using menotropins) with intrauterine insemination without pregnancy, now undergoing In Vitro Fertilization (for unexplained infertility). On the day of oocyte (egg) retrieval, 8 eggs were obtained and transferred to the embryology laboratory were they were inseminated with sperm (50,000 motile sperm per egg within a microdroplet of media). On the day following egg retrieval, none of the eggs showed evidence of fertilization.

Question: What should be considered given this information?

Answer: It is impossible to predict with absolute certainty which sperm will fertilize eggs at the time of IVF. Generally, there is an approximate 70-80% fertilization rate with standard microdroplet insemination of eggs at IVF if the sperm quality looks normal. However, on occasion there will be no fertilization identified the day after insemination even when the sperm appears totally normal. This is upsetting for everyone involved. Assisted fertilization (ICSI) can be attempted on these “day old” eggs yet the success rates (in terms of pregnancy) are relatively low.

Given that it is not possible to be certain of fertilization the day following IVF oocyte retrieval with standard insemination techniques, some IVF programs recommend assisted fertilization (ICSI) as a way to avoid cases of “unexpected” lack of fertilization. ICSI may be suggested relatively liberally whenever there is any abnormality in the semen analysis, a thickened or hardened egg zona pellucida (shell), unproven fertility for the man (no history of prior pregnancy), or a lot of eggs retrieved (eg., if 20 eggs are retrieved then it is possible to split the eggs into 2 groups of 10 eggs with one group having standard insemination and the other group having ICSI).

Case: 34 year old G0 with a history of subfertility, regular menstrual intervals every 25-26 x 2-3 days, a normal hormone evaluation (TSH and prolactin), an encouraging ovarian reserve (using basal FSH and estradiol concentrations), a normal hysterosalpingogram (normal uterine cavity with bilateral tubal patency), a normal postcoital test, a husband with proven fertility (two children in a prior marriage) and a normal semen analysis, status post pelvic evaluation (laparoscopy and hysteroscopy) that did not reveal any abnormalities, status post 4 cycles of controlled ovarian hyperstimulation (using menotropins) with intrauterine insemination without pregnancy, now undergoing In Vitro Fertilization (for unexplained infertility). On the day of oocyte (egg) retrieval, 14 eggs were obtained and transferred to the embryology laboratory were they were inseminated with sperm (50,000 motile sperm per egg within a microdroplet of media). On the day following egg retrieval, 8 of the 14 eggs had 3 pronuclei (showed evidence of fertilization with more than one sperm), 4 of the eggs had 2 pronuclei (appeared to be fertilized normally) and 2 of the eggs did not fertilize.

Question: What should be considered given this information?

Answer: Normally, only one sperm is allowed to fertilize an egg. At the time of fertilization, the sperm normally releases its genetic material (chromosomes) into the cytoplasm of the egg and the egg subsequently undergoes a “cortical reaction” (series of molecular events) that blocks further penetration by other sperm. If more than one sperm is allowed to fertilize the egg, there is an abnormal amount of chromosomal material within the egg (resulting in a lethal mutation).

Polyspermic fertilization (fertilization with more than one sperm) is uncommon and usually does not present a clinical problem. Generally, at IVF less than 10% of fertilized eggs have three or more pronuclei. In this situation, an unusually high percentage (75%) of the fertilized eggs (8 of the 12 eggs that were actually fertilized by the sperm) contained three pronuclei. When such a high percent of the fertilized eggs have had a major problem with fertilization, the remaining normally fertilized eggs tend to do less well (than otherwise expected) and the underlying problem is often thought to rest with the eggs.

If the woman in this example underwent a standard (long) menotropin protocol (with lupron beginning around 7 days after ovulation and prior to the onset of the next menstrual flow) and has abnormal appearing embryos (such as a high percentage of polypronuclear embryos) then I often suggest the microflare protocol for subsequent cycles of IVF (if needed). I have had relatively surprising success (very high percentage of pregnancies at IVF) using the microflare protocols for women in this situation.

Case: 38 year old G4 P4 status post bilateral tubal ligation (had her tubes tied) now remarried (and seeking fertility) with a history of regular menstrual intervals every 30 x 4-5 days, a normal hormone evaluation (TSH and prolactin), an encouraging ovarian reserve (using basal FSH and estradiol concentrations), a normal uterine cavity on hysterosalpingogram (with bilateral tubal occlusion), a husband with unproven fertility and an abnormal semen analysis (oligospermia with a slightly low concentrations ranging from 8-15 million sperm per mL), status post pelvic evaluation (laparoscopy and hysteroscopy) that did not reveal any abnormalities, now undergoing In Vitro Fertilization (for tubal factor infertility). At egg retrieval 9 oocytes (eggs) are obtained and immediately transferred to the embryology laboratory.

Question: What should be considered given this information?

Answer: In this situation there is unproven (male) fertility with a suggestion of a mild male factor. IVF is required for reproduction since the woman has had her tubes tied (bilateral tubal ligation) after 4 deliveries in a prior marriage and does not want a tubal reanastomosis (reversal of the tubal ligation).

I would generally suggest assisted fertilization (ICSI) for all of the eggs that are retrieved at IVF since it is impossible to reliably predict whether the sperm will function normally (fertilize 70-80% of eggs) with standard microdroplet insemination. Since IVF will be required for all attempted reproduction in this relationship the need to see whether this sperm fertilizes eggs on their own (without assisted fertilization) is of limited utility.

Embryo Development

Case: 26 year old G0 with a history of regular menstrual intervals every 28-30 x 3-5 days, a normal hormone evaluation (TSH and prolactin), an encouraging ovarian reserve (using basal FSH and estradiol concentrations), a normal hysterosalpingogram (normal uterine cavity with bilateral tubal patency), a normal postcoital test, a husband with unproven fertility and a normal semen analysis, status post pelvic evaluation (laparoscopy and hysteroscopy) that did not reveal any abnormalities, status post 4 cycles of controlled ovarian hyperstimulation (using menotropins) with intrauterine insemination without pregnancy, now undergoing In Vitro Fertilization (for unexplained infertility).

16 mature eggs are obtained on the day of oocyte (egg) retrieval, transferred to the embryology laboratory, and incubated with sperm (50,000 motile sperm per egg within a microdroplet of media). On the day following egg retrieval there was apparently normal fertilization of 13 of the 16 eggs. Two days after egg retrieval all 13 fertilized eggs (pre-implantation embryos) contained 2-4 cells. Three days after egg retrieval 10 of the 13 fertilized eggs progressed to contain 6-10 cells.

Question: What should be considered given this information?

Answer: Until about 1999, most embryo transfers at the time of In Vitro Fertilization (IVF) took place on the 2nd or 3rd day following egg retrieval. In 2001, most embryo transfers seem to occur on the 5th day following egg retrieval when the fertilized eggs are at the “blastocyst” stage of development.

Until recently, embryologists were not able to “push” embryos until the 5th day after retrieval with the consistent development of blastocyst stage embryos. Many of the developing embryos would arrest in development after the 3rd or 4th day in culture. Therefore, if the embryos were not transferred into the uterine cavity and they all arrested (in development) in culture then there would be no viable embryos to replace.

Currently, most of the In Vitro Fertilization laboratories that I am familiar with have the ability to culture embryos to the 5th post retrieval day at which point they are consistently late stage morulas or blastocysts. Embryo transfer using embryos at the blastocyst stage of development has the theoretical advantage of replacing embryos into the uterus at the same stage of development that they would normally enter the cavity in a natural cycle (if fertilization took place spontaneously within the fallopian tube rather than through IVF). IVF with embryo transfer of blastocysts has a much greater pregnancy rate (per embryo transfered) than IVF with embryo transfer of post retrieval day 2-3 embryos. Therefore, I generally only place 2 blastocysts into the uterine cavity. With embryo transfer on post egg retrieval day 2-3 generally 4 good quality embryos are placed into the uterine cavity.

I usually suggest allowing the embryos to develop to the blastocyst stage (or post retrieval day 5) when there are greater than 5 good looking embryos growing on post retrieval day 2-3. In the case here, there were 10 embryos on post egg retrieval day 3 so I would usually suggest a day 5 transfer in this situation.

Case: 37 year old G0 with a history of regular menstrual intervals every 27-29 x 3-5 days, a normal hormone evaluation (TSH and prolactin), an encouraging ovarian reserve (using basal FSH and estradiol concentrations), a normal hysterosalpingogram (normal uterine cavity with bilateral tubal patency), a normal postcoital test, a husband with unproven fertility and severe oligospermia on semen analysis (3-5 million sperm per mL), status post pelvic evaluation (laparoscopy and hysteroscopy) that did not reveal any abnormalities, now undergoing In Vitro Fertilization with assisted fertilization (ICSI) for male factor infertility.

13 mature eggs are obtained on the day of oocyte (egg) retrieval, transferred to the embryology laboratory, and assisted fertilization using ICSI resulted in 10 normal appearing fertilized eggs on the day following egg retrieval. Two days after egg retrieval 7 of the 10 fertilized eggs (pre-implantation embryos) continued to divide (grow) and contained 2-4 cells. There was a significant difference in the sizes of the cells (blastomeres), extensive fragmentation, and intracellular vacuolization.

Question: What should be considered given this information?

Answer: I generally would allow good quality embryos to develop for 5 days after egg retrieval (prior to embryo transfer into the uterine cavity) since the reproductive potential of the embryos is greater (therefore the number of embryos that are transferred can be lower without sacrificing pregnancy rates).

If less than 5-6 good looking embryos are developing on post retrieval days 2-3 then there appears to be a significantly greater chance of having no embryos for transfer if they are all “pushed” to day 5. In this case, there are 7 embryos that are actively growing on post egg retrieval day 2 but the quality of the embryos (as described) is poor. At this point, the decision needs to be whether to return several of the developing embryos into the uterus on post retrieval day 2-3 or whether to wait until post retrieval day 5.

Embryologists and Reproductive Endocrinologists have various opinions on this issue.

Some think that if the embryos are of good enough quality to result in a pregnancy, then they will also be of good enough quality to survive until post egg retrieval day 5. If this is correct, then those embryos that arrest in development prior to day 5 would also not have resulted in a pregnancy (so that if none of the embryos survive to the 5th day then you are essentially saving the couple an embryo transfer procedure, several weeks of progesterone in oil injections, and 2 weeks of anxiety wondering whether the cycle may be successful).

Others believe that the nonphysiologic environment of the IVF laboratory (in which the embryos are growing) presents a “stress test” for embryos, such that some embryos will grow abnormally in this environment (in the incubator of a laboratory) but may be able to be “rescued” such that they will grow normally if placed into the “friendlier” environment of the uterine cavity. If this is correct, then some of the embryos that do not look good on day 2-3 after retrieval may perform better in the uterus and may result in normal pregnancies (so that an embryo transfer on day 2-3 after retrieval is worthwhile).

I generally inform my patients about the appearance of the embryos on day 2-3 and review (with their input) whether an embryo transfer on day 3 or day 5 would be better. Poor embryo quality is most often believed to be due to poor egg quality rather than the environment in which the embryos are growing. However, many of my patients with poor embryo quality have undergone embryo transfer on post egg retrieval day 3 (since we did not think that the embryos would survive to day 5) and did become pregnant with subsequent normal embryonic and fetal development. Of course, I don’t know how these embryos would have progressed in culture if they were not transferred on (postretrieval) day 3. Therefore, I will continue to offer this as an option until more is known in this important area of research.

Case: 23 year old G0 with a history of regular menstrual intervals every 30-32 x 4-6 days, a normal hormone evaluation (TSH and prolactin), an encouraging ovarian reserve (using basal FSH and estradiol concentrations), a normal hysterosalpingogram (normal uterine cavity with bilateral tubal patency), a normal postcoital test, a husband with unproven fertility and a normal semen analysis, status post pelvic evaluation (laparoscopy and hysteroscopy) that did not reveal any abnormalities, now undergoing In Vitro Fertilization (for unexplained infertility).

27 mature eggs are obtained on the day of oocyte (egg) retrieval. These eggs were transferred to the embryology laboratory and they were incubated with sperm (50,000 motile sperm per egg within a microdroplet of media). On the day following egg retrieval there was apparently normal fertilization of 25 of the 27 eggs. Two days after retrieval the 25 fertilized eggs (pre-implantation embryos) contained 2-4 cells. Three days after egg retrieval 22 of the 25 fertilized eggs progressed to contain 6-10 cells. Five days after egg retrieval 16 of the 22 embryos continued to grow and yet all of these were in the morula rather than the blastocyst stage of development.

Question: What should be considered given this information?

Answer: By the 5th day following egg retrieval most of the developing embryos are normally at the blastocyst stage of development. Morulas may represent a mild developmental delay in the embryos that is of unclear clinical significance. Usually, if the day 5 embryos are all morulas then one difficult decision becomes how many embryos to return to the uterus.

If there are only morulas to transfer to the uterine cavity on the 5th day after egg retrieval, I generally have suggested transfer of 3 (rather than 2) embryos. My impression is that if the embryos are morulas on day 5 then they have a reduced reproductive potential (suggested by the “minor” delay in their development) compared to blastocysts. However, when 3 embryos are transferred there is a significant chance of triplets so I discuss this with the couple undergoing the procedures and we factor in (to the decision of whether to place 2 or 3 embryos at transfer) their feelings with regard to selective reduction versus carrying a triplet pregnancy.

15 mature eggs are obtained on the day of oocyte (egg) retrieval. These eggs were transferred to the embryology laboratory and they were incubated with sperm (50,000 motile sperm per egg within a microdroplet of media). On the day following egg retrieval there was apparently normal fertilization of only 7 of the 15 eggs. Two days after retrieval the 7 fertilized eggs (pre-implantation embryos) contained 2-6 cells but significant fragmentation was noted. Three days after egg retrieval only 4 of the 7 fertilized eggs progressed to contain 6-10 cells and the grading of the embryos was disappointing (poor).

Question: What should be considered given this information?

Answer: Poor embryo quality (as judged by the embryologists who grade the them) often results in unsuccessful IVF cycles. Unfortunately, a very good IVF candidate with a good response to controlled ovarian hyperstimulation (COH) can have poor embryo quality even at one of the nation’s leading IVF centers. If the IVF cycle does not result in a pregnancy then it is difficult to predict whether a repeat IVF cycle will have a similar or a much more favorable outcome.

The cause of poor embryo quality is generally thought to lie in the oocyte (egg) rather than the sperm. The exact nature of the abnormality rarely is discovered, but may involve genetics (abnormal nuclear DNA content possibly due to errors in the segregation of the chromosomes during meiosis) or the intrinsic reproductive potential of the egg (cytoplasmic contents of the egg include structural proteins, regulatory proteins, and other intracellular molecular messengers that may be prone to malfunction).

In these situations, if the initial IVF cycle resulted in surprisingly poor embryo quality I suggest a 2nd cycle of IVF.

The same COH stimulation protocol has often been used at major IVF centers and even when the same protocol is used with the same woman the outcome in two different IVF cycles can be vastly different. This difference in outcome (in terms of both embryo quality and reproductive success) may be due to the recruitment of totally different cohorts of eggs from the ovaries.

If the embryo quality during an IVF cycle is unexpectedly poor after using a standard (long suppression) protocol (with lupron started about 7 days prior to expected menses) then I try another IVF cycle but I generally switch to a microflare lupron protocol since I have seen good results in these situations with this protocol. The number of cycles of successful IVF outcomes using a microflare protocol after a prior unsuccessful IVF cycle using a standard protocol is in my experience enough to suggest this to my own patients at least until the large research IVF centers come up with a better (more successful) suggestion.

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