Luteinizing hormone and ovulation timing in a therapeutic donor insemination program using frozen semen

Luteinizing hormone and ovulation timing in a therapeutic donor insemination program using frozen semen Liliana R. Kossoy, MD: George A. Hill, MD: Robert A. Parker, DSc; B. Jane Rogers, PhD: Carol S. Dalglish, RN: Garl M. Herbert III, MD: and Anne Colston Wentz, MD" Nashville, Tennessee A series of 110 therapeutic donor insemination cycles was analyzed to determine the impact on fecundity when a urinary luteinizing hormone detection kit was used to plan inseminations. To prevent the transmission of human immunodeficiency virus, frozen semen, thawed after a 90-day quarantine, was used. The minimum standard for insemination with cryopreserved semen was a total of 24 x 10S motile sperm per milliliter after thawing. Fecundity was 0.12 when insemination timing was based on cervical mucus evaluation and basal body temperature charts and 0.13 when a urinary luteinizing hormone kit was also used to predict ovulation. Life table analysis with the log rank test showed no statistically significant difference in the number of cycles required to achieve conception betwen the group of patients using conventional methods of ovulation timing and the group of patients using the urinary luteinizing hormone kit. Urinary luteinizing hormone testing offers no advantage over conventional methods, such as cervical mucus examination and evaluation of basal body temperature, when ovulation is being timed for insemination with frozen donor semen. (AM J OasTET GVNECOL 1989;160:1169-72.)

Key words: Therapeutic donor insemination, urinary luteinizing hormone, frozen semen

In response to the concern regarding the possible transmission of human immunodeficiency virus when fresh semen is used for therapeutic donor insemination,l our program began to freeze and quarantine all semen samples. After thawing, spermatozoa show a lower rate of survival and a higher frequency of loss of acrosomal integrit y2 and other intracellular functions'; therefore ovulation timing to plan inseminations might become critical to compensate for latent cryoinjury" In a previous study,' the use of an enzyme immunoassay of urinary luteinizing hormone (LH) to predict ovulation in addition to basal body temperature (BBT) charts and cervical mucus examinations to plan inseminations did not reduce the number of cycles required to achieve conception in a population receiving therapeutic donor insemination with fresh semen. The purpose of this study was to determine the effect of the use of a urinary LH detection kit to predict ovulation and time inseminations on the monthly fecundity rate in a therapeutic donor insemination program using frozen and thawed semen. From the Center for Fertilzty and Reproductive Research (C-FARR), Department of Obstetrics and Gynecology," and the Department of Preventive Medicine, VanderbIlt University Medical Center. h Presented at the Fortyjourth Annual Meeting of the Amencan Fertility Society, Atlanta, Georgia, October 8-13, 1988. ReceIVed for publication july 5, 1988; reVISed November 28, 1988; accepted December 15, 1988. Repnnt requests: George A. Hill, MD, Center for Fertility and ReproductIVe Research, Department of Obstetrics and Gynecology, Rm. D-3223, Vanderbilt UniverSIty MedIcal Center North, Nashville, TN 37232.

Material and methods The study population consisted of 54 patients undergoing therapeutic donor insemination with frozen and thawed semen betwen October 1987 and February 1988. During that time, specimens were released 90 days after freezing when the donor was retested and negative results found in the serum for human immunodeficiency virus. Indications for therapeutic donor insemination in these patients were azoospermia (n = 27) and severe oligospermia (n = 27). Ovulation problems and luteal phase inadequacy were recognized and corrected before and during cycles of insemination. In patients who had never had uterine, tubal, and peritoneal factors evaluated, hysterosalpingography and laparoscopy were performed after three and six failed insemination cycles, respectively. The approximate day of ovulation was calculated by studying the BBT chart obtained in past menstrual cycles. Therapeutic donor insemination was performed in the presence of adequate cervical mucus evaluation results (clear, profuse mucus, with maximum ferning and spinnbarkeit), provided the BBT had not shown an upward shift. All patients were offered the option of using urinary LH detection in addition to BBT and cervical mucus examinations to plan inseminations. Patients who used urinary LH detection (group 1, n = 31) (OvuStick, Monoclonal Antibodies Inc., Mountain View, Calif.) checked the urine at home, once a day, starting 2 days before the expected day of the LH surge. Inseminations were done when the color obtained with the test was 1169

1170 Kossoy et al.

May 1989 Am J Obstet Gynecol

5040~

Z

~ 30C!)

ILl

i

0::

a.. 20;I. 10-

o

r----

I

I

~

2

--Group 1 ---Group 2

CYCLES

3

Fig. 1. Cumulative fecundity rates for groups I and 2. Cycles, Cycles

equal to or greater than the surge guide. Patients in group 2 (n = 23) did not use urinary LH testing. Only BBT and cervical mucus examinations were used to plan inseminations in these patients. Each patient received one or two inseminations per cycle with a fitted Mylex cervical cup (Milex Products, Inc., Chicago). The cup was placed on the cervix in the office and was removed by the patient in 6 to 8 hours. Sperm-cervical mucus interaction was evaluated during the first cycle of insemination. Freezing protocol. Donors are selected for the artificial insemination program on the basis of medical history and semen characteristics. To be included in the donor program, the screening semen sample must have at least 80 million spermatozoa per milliliter and 60% motility. After thawing, the sample must have at least 12 million motile sperm per milliliter. If these criteria are met, further screening includes serum tests for human immunodeficiency virus, syphilis, hepatitis B surface antigen and semen tests for chlamydia, gonorrhea, and white blood cells. The semen sample is allowed to liquety tor 3U mmutes at room temperature before being mixed with an equal volume of cryoprotectant medium containing egg yolk (20%), calcium chloride (7 mmoIlL), magnesium chloride (4.8 mmoIlL), zinc chloride (2.3 mmoIlL), glycine (267 mmoIlL), streptomycin (500 mg/ml), and penicillin G (1000 units/ml). The samplecryoprotectant mixture is placed by aliquot into 1 ml cryovials with screw caps. The specimens to be frozen are suspended in liquid nitrogen vapors for 30 minutes before storage in liquid nitrogen. To thaw a sample, the specimen is removed from liquid nitrogen and placed in a 37° C water bath for 5 minutes. Insemination is performed with two vials of thawed semen, which is equivalent to a 2 ml volume and at least 24 million motile sperm. Statistical analysis. Life table analysis and the log

5

4 to

pregnancy.

rank test were used to compare outcomes between groups. The starting point in the life table analysis was the first insemination cycle. End points were the first conception in a patient, crossover between groups, and the end of the study.6 Fisher's exact test was used to compare the proportion of factors affecting fertility in both groups. Statistical significance was defined as p < 0.05.

Results Pregnancies occurred in 13 patients. Monthly fecundity for the study cycles is presented in Fig. 1. In group 1, nine pregnancies were achieved during 67 insemination cycles, for an average fecundity of 0.13. In group 2, five pregnancies were achieved during 43 insemination cycles, for an average fecundity of 0.12. Life table analysis with the log rank test showed no statistically significant difference between groups when the number of insemination cycles until conception was compared: p> 0.05 (Fig. 1). Table I shows the cumulative probability of conception for both groups. Patients in group 1 received 1.6 ± 0.7 (mean ± SD) inseminations per cycle, and patients in group 2 received 1.5 ± 0.5 (mean ± SD) inseminations per cycle. The fertility-related factors analyzed (age >30, husband with oligospermia versus azoospermia, adhesions or endometriosis diagnosed at laparoscopy, or need for ovulation induction) were not statistically significantly different when both groups were compared (Table II).

Comment In the American Fertility Society'S new guidelines for the use of donor insemination, the use of fresh semen is no longer warranted and a quarantine of 180 days is recommended for all frozen specimens. 7 To provide maximum safety for patients undergoing therapeutic donor insemination, cryopreserved specimens have been substituted for fresh semen in our program. The

Frozen donor semen and ovulation timing

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Table I. Number of patients entering each insemination cycle, pregnancies, and cumulative probability of conception for groups 1 and 2 E ntenng cycle Cycle no.

Group 1

1 2 3 4 5 Total

31 22 10 3 1 31

I

Cumulative probabllzty of conception (%)

ConceIved

Group 2

Group 1

23 10 6 2 2 23

I

I

Group 2

Group 1

3 4 2

4 1

9.7 26.1 40.9

17.4 25.7 25.7

9

5

40.9

25.7

Group 2

Table II. Proportion of patients in each group with associated infertility factors (Fisher's exact tests [two-sided ]) Vanable

Group 1 (%)

Group 2 (%)

p value

Age >30 yr Need for ovulation induction Positive laparoscopic findings Husband oligospermic

43 39 48 58

30 39 30 39

NS NS NS NS

initial 90-day quarantine reported in this work has been extended to 180 days since the new guidelines were published in February 1988. Cryopreserved sperm is thought to be less efficient than fresh ejaculated sperm in producing pregnancies."·10 As a result of the freezing and thawing process, a significant decrease in motility has been shown, II as well as a decrease in several intracellular functions' and a higher frequency of loss of acrosomal integrity.2 Frozen and thawed sperm have also been shown to have a lower frequency of survival. 2. 12. 13 If frozen and thawed spermatozoa have a reduced longevity in the female genital tract as shown in vitro, their use in therapeutic donor insemination might not always provide viable sperm in the upper genital tract at the time of ovulation. Thus, provided an acceptable cryopreservation technique is used and high standards for motility after thawing are kept, insemination timing might become decisive" According to Bordson et al.,14 the poor fecundity rates experienced by clinics using frozen semen might be related to inadequate minimum criteria used for postthaw insemination samples. These authors found no significant difference in fecundity rates when frozen rather than fresh semen was used (0.10 versus 0.12). Inseminations in their program were performed after the injection of human chorionic gonadotropin in stimulated cycles or after the natural urinary LH surge as determined by urinary LH testing. II Fecundity rates in our therapeutic donor insemination program' have not dropped since cryopreserved semen specimens with high minimum criteria for motility after thawing (12 million motile sperm per milliliter) have been substituted for fresh semen. The long

survival of sperm in estrogenic cervical mucus l5 may account for the lack of a significant difference in monthly fecundity rates between patients using urinary LH testing and those using only BBT and cervical mucus examinations to plan inseminations with fresh donor semen.' The present study shows that when frozenlthawed semen is used, insemination timing with the aid of urinary LH detection to predict ovulation does not improve fecundity over that obtained when traditional methods of ovulation timing (BBT, cervical mucus examinations) are used. Because pregnancy itself is the only true measure of sperm functional capacity, it can be suggested that with strict standards for motility after thawing, a highly qualified sub population of resistent spermatozoa will be selected that has a life span that will overlap ovulation. Our results suggest that although urinary LH detection adds to traditional methods of ovulation timing, BBT and cervical mucus examinations are still valuable indicators of ovulation and can be used as single methods to time inseminations in therapeutic donor insemination programs that use frozen and thawed specimens. REFERENCES 1. Stewart Gj, Tyler jPP, Cunningham AL, et a\. Transmission of human T-celllymphotropic virus type III (HTLV III) by donor. Lancet 1985;2:581. 2. Crister jK, Huse-Bonda AR, Aaker DJ. Arneson BW, Ball DG. Cryopreservation of human spermatozoa. I. Effects of holding procedure and seeding on motility, fertilizability, and acrosome reaction. Fertil Steril 1987; 47:656. 3. Ackerman DR. Fructose utilization of human spermatozoa after cooling and freezing. lut J Ferti! 1967; 12: 1. 4. Sherman JK. Cryopreservation of spermatozoa. In: Lu-

Kossoy et al.

5.

6. 7. 8. 9.

nenfeld B, Insler V, eds. Infertility: male and female. Edinburg: Churchill Livingstone, 1986:590. Kossoy LR, Hill GA, Herbert CM, et al. Therapeutic donor insemination: the impact of insemination timing with the aid of a urinary luteinizing hormone immunoassay. Ferti! steril 1988;49: 1026. Cramer DW, Walker AM, Schiff I. Statistical methods of evaluating the outcome of infertility therapy. Fertil Steril 1979;32:80. The American Fertility Society. Revised new guidelines for the use of semen donor inseminations. Ferti! Steril 1988;49:211. Leeton], Selwood T. Trounson A. Wood C. Artificial donor insemination: fresh versus frozen semen. Aust N Z] Obstet Gynaecol 1980;20:205. Smith KD, Rodriguez-Rigau L]. Steinberg E. The influence of ovulatory dysfunction and timing of insemination on the success of artificial insemination donor (AID) with fresh or cryopreserved semen. Ferti! Steri! 1981 ;36:496.

May 1989 Am J Obstet Gynecol

10. Richter MA, Haning RV]r, Shapiro SS. Artificial donor insemination: fresh versus frozen semen, the patient as her own control. Ferti! Steril 1984;41 :277. 11. Thatchil]V,]ewett MAS. Preservation techniques for human semen. Ferti! SteriI1981;35;546. 12. Keel BA, Black]B. Reduced motility longevity in thawed human spermatozoa. Arch Androl 1980;4:213. 13. Ulstein M. Fertility, motility, and penetration of cervical mucus of frozen-preserved human spermatozoa. Acta Obstet Gynecol Scand 1973;52:205. 14. Bordson BL, Ricci E, Dickey RP, Dunaway H, Taylor SN, Curole DN. Comparison of fecundability with fresh and frozen semen in therapeutic donor insemination. Fertil Steril 1986;46:466. 15. Moghisi KS. Cyclic changes of cervical mucus in normal and progestin treated women. Ferti! Steri! 1966; 17:663.

Preterm birth prevention: Evaluation of a prospective controlled randomized trial Eberhard Mueller-Heubach, MD, Debra Reddick, RN, Barbara Barnett, LPN, and Robert Bente Ptttsburgh, Pennsylvania Over a 3-year period 5457 indigent patients were scored for risk of preterm birth and 4595 women were delivered at 2:20 weeks' gestation. Patients at high risk (18.1 %) were randomized into control and intervention groups. The latter group received weekly cervical examinations and instruction regarding subtle symptoms and signs of preterm labor. Medical providers received similar instruction. There was no difference in preterm births between control and intervention groups (20.8% vs. 22.1%). Medical providers, convinced of preterm birth prevention during year 1 of the study, defeated the study design by giving preterm birth precautions to all patients. In turn, preterm births decreased from 13.7% (year 1) to 9.3% (year 2, p < 0.001) and remained stable in year 3 (8.7%). Preterm births during year 1 and the 8 months preceding year 1 were not different. Significant differences in preterm births between private and indigent study patients during these two periods (p < 0.001) disappeared during years 2 and 3 of the study. (AM J OSSTET GVNECOL 1989;160:1172-8.)

Key words: Prematurity, preterm labor, premature rupture of membranes, tocolysis Preterm birth is the major factor responsible for differences in infant mortality in industrialized nations. There is an increased incidence of neurodevelopmental handicaps, chronic respiratory problems, and retrolental fibroplasia in infants who survive pre term birth. Prolonged hospitalization of the preterm infant in neoFrom the Department of Obstetrics and Gynecology, UnlVemty of Pittsburgh School of MediCine. Magee-Womens Hospital. Supported by March of Dimes Grant 2-196/C-404. Received for publzcation May 3, 1988; revised October 15, 1988; accepted December 1, 1988. Reprint requests: Eberhard Mueller-Heubach, MD, Magee-Womens Hospital, Forbes Ave. and Halket St, Pittsburgh, PA 15213.

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natal intensive care units, frequent rehospitalization, and need for long-term care make preterm birth an extremely expensive issue and a major aspect of health care expenditure.' Recognition ot the;e problems related to preterm birth has led to considerable interest in identifying pregnant women who are at high risk of preterm birth and attempts at pre term birth prevention in this population. Reports of preterm birth prevention projects in this country; in Martinique,' and in France 4 suggest that it is possible to decrease the preterm birth rate. However, these studies are of limited scientific value because of their design. None of them were performed